Naghibione; A Novel Sesquiterpenoid with Antiplasmodial Effect from Dorema hyrcanum Koso-Pol. Root, a Plant Used in Traditional Medicine

Some Dorema species are used in Persian traditional medicine. In the present study the total extract from the roots of Dorema hyrcanum Koso-Pol. was investigated for its in-vitro (pLDH assay) and in-vivo (Peters’ 4-days suppressive test) antiplasmodial effects and assessed for cytotoxicity against the normal cell line MDBK (MTT test). The IC50 values for a chloroquine- sensitive (3D7) and a chloroquine- resistant (K1) strain of Plasmodium falciparum were 28.64 and 9.79 µg/mL, respectively. The inhibition percentage of the rodent parasite, Plasmodium berghei, on day 4 in mice was 77.9% and IC50 value on Madin–Darby bovine kidney cells (MDBK cells) was 59.84 µg/mL. The total extract was subjected to a bioassay-guided fractionation protocol based on the in-vivo model which resulted in the isolation of an acetophenon (compound 1), one new sesquiterpenoid; naghibione (compound 2) and two known sesquiterpenoid derivatives (compounds 3, 4). Their structures were elucidated by spectroscopic analysis, including 1D and 2D NMR experiments and ESI-MS. All compounds were evaluated for in-vivo antiplasmodial effect and the results revealed that naghibione showed good suppression activity, inhibiting 68.1 % of the parasite growth.


Introduction
Malaria is a global public health problem whose main victims are children under five years of age in Africa. According to the latest WHO estimates, there were about 219 million cases of malaria in 2010. and an estimated 660000 deaths(1).
Antimalarial drug resistance is a major concern for the global effort to control malaria. P. falciparum resistance to artemisinin has been detected in four countries in South East Asia: in Cambodia, Myanmar, Thailand and Vietnam. There is an urgent need to expand containment efforts in affected countries(1).
Traditional medicine is a potential rich source of new drugs against malaria and other infectious diseases. It has made remarkable contributions to the development of potent antimalarial drugs (2).

Plant material
The roots of D. hyrcanum (Apiaceae), collected in June 2008 from Almed Valley in Golestan District, Iran, were identified by Mr. H. Moazeni from Traditional Medicine and Materia Medica Center (TMRC), Shahid Beheshti University of Medical Sciences, Iran. The voucher specimen 2495 (TMRC) of the plant has been deposited in the herbarium of the TMRC.

Extraction
The powdered dried root of D. hyrcanum was macerated in methanol (MeOH), for 24 hours with constant shaking, at room temperature. The filtrates of total extract were evaporated to dryness and investigated for its in-vitro and invivo antiplasmodial and cytotoxic effects.

Biological assays
In-vitro antiplasmodial activity Antiplasmodial activity of the total extract was determined against the chloroquineresistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum that were continuously cultured according to the methods described by Trager and Jensen (13). Plant extract was assessed for antiplasmodial activity in-vitro in human blood using parasite lactate dehydrogenase method (pLDH) with slight modifications(14, 15). The range of examined concentration was from 64 µg/mL to 125 ng/ mL. A solution of chloroquine diphosphate and artemisinin served as positive control. The test was performed in duplicate. Absorbance was measured with an ELISA plate reader at 630 nm. The percentage inhibition at each concentration was determined and the mean of IC 50 value of parasite viability was calculated using Probit analysis(16).

In-vivo antimalarial assay
Peters' 4-day suppressive test against NICD strain of Plasmodium berghei infection in mice was employed(17) for evaluating of the samples of D.hyrcanum antiplasmodial activities. Adult male albino mice from the Pasteur Institute of Iran were housed under standard environmental conditions and fed with standard pellets and water. All the procedure was accepted by Shahid Beheshti University of Medical Sciences Seven species of the Dorema (Apiaceae) are included in the flora of Iran(3). In Iran, Dorema species with the local names "Oshagh" or "Vosha"(4) are used in traditional medicine for different purposes such as pesticide, antihelminthic, expectorant and also for treatment of spleen and liver stiffness (5).
According to literature reviews, antimicrobial activity of D. ammoniacum(6) and the presence of weak antioxidant and antihemolytic activities in a hydroalcoholic extract of D. aitchisonii have been reported(7). D. aucheri is the first umbelliferous plant found to produce exudate flavonoids(8). Three sesquiterpene derivatives, one prenylated coumarin and two steroid glucosides were isolated from the aerial parts of D. kopetdaghense(9). There is a report about the phytochemical constituents of D.hyrcanum that mentions two glycosides, namely pleoside and hyrcanoside(10) and in an initial screening for anti-tumor agents by an in-vitro microbiological assay D. hyrcanum did not induce phage production(11). According to our studies there have been no scientific reports regarding antiplasmodial effect of these species.
In this search, for the first time, D. hyrcanum Koso-Pol., which is endemic to Iran, Turkmenistan and Afghanestan(12), has been investigated for its antiplasmodial effect by invitro and in-vivo assays and evaluated for its invitro cytotoxicity. A bio-assay guided isolation led to the identification of four compounds.

General experimental procedures and materials
NMR spectra were recorded on a Bruker DRX 500 spectrometer. Chemical shifts are given relative to TMS as an internal standard. ESI mass spectra were carried out on an Agilent 6210 ESI-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Semi preparative HPLC was performed on a Shimadzu instrument with PRC-ODS C18 (20 mm × 25 cm) column. Silica gel 60 (0.063-0.2; 0.2-0.5 mm; Merck) was used for column chromatography. All solvents for semi preparative HPLC were of technical grade and purified by distillation.
Ethics Committee and in accordance with the principles for laboratory animal use and care in the European Community guidelines.
Briefly, the parasites (blood contained parasites) were maintained by serial passage of blood from mouse to mouse. Adult male albino mice weighing 20-25 g were inoculated by intra-peritoneal (I.P) injection with 1×10 7 infected erythrocytes. The mice were randomly divided into groups of five per cage and treated during consecutive days with 10 mg/mL of the sample by I.P injection for 4 days. Two control groups were used in this experiment, one treated with chloroquine at dose of 20 mg/ Kg as a positive control while the other group was kept untreated as a negative group. On day 5 of the test, thin blood smears were prepared and blood films were fixed with methanol. The blood films were stained with Giemsa, and then microscopically examined. Percentage of parasitaemia was counted based on infected erythrocytes calculated per 1000 erythrocytes and the inhibition percentage of each group expressed in relation to the untreated group.
In-vitro cytotoxicity assay Cytotoxicity of samples was determined using MDBK cells(2, 18) by the colorimetric methyl thiazole tetrazolium (MTT) assay (19,20) and scored as a percentage of absorbance reduction at 570 nm of treated cultures versus untreated control cultures. Tests were run in triplicate and tamoxifen was used as a positive control. IC 50 values were calculated from the drug concentration-response curves.

Results and Discussion
In this study for the first time D. hyrcanum was evaluated for its antiplasmodial activity. In a screening program, its methanolic extract was tested against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) as well as a chloroquine-resistant strain (K1) and showed IC 50 values of 28.64 and 9.79 µg/ mL, respectively. Furthermore it was evaluated by Peters' 4-day suppressive test and revealed 77.9% inhibition of P. berghei on day 4 in mice at a concentration of 10 mg/Kg. In a cytotoxicity assay against MDBK cells, it showed an IC 50 value of 59.84 µg/mL.
A bioassay-guided fractionation protocol based on the in-vivo model (Peters' test) was assessed in order to isolate the active compounds. Therefore, the ethyl acetate extract, some of its selected fractions and pure compounds were evaluated for antiplasmodial activity by the invivo test and for cytotoxicity (Table 1).
Fractions 2, 3 and 4 were exhibited higher antiplasmodial activity and lower cytotoxicity than that of the total extract. All pure compounds were evaluated for in-vivo antiplasmodial effect and the results revealed that compound 2 showed good suppression activity, inhibiting 68.1 % of the parasite growth.

Conclusion
In this study for the first time, Dorema hyrcanum Koso-Pol. was evaluated for its antiplasmodial activity and naghibione (compound 2); a new sesquiterpenoid with antiplasmodial activity has been isolated. Furthermore, two known sesquiterpenoids 3 and 4 were for the first time isolated from this genus.
Further studies in order to evaluate the mode of action of the pure compounds are under way.

Acknowledgement
This paper is a part of Traditional Pharmacy Ph.D thesis (122) conducted by Saeedeh Ghafari, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences. Also this research was supported by two Grants (No. 115,No. 7445) from Shahid Beheshti University of Medical Sciences.
A congener with a 4-hydroxy group instead of the methoxy group was obtained from Ferula ferulaeoides (22) and with a carbonyl group on C9 from Dorema kopetdaghense (9) previously. In the best our knowledge the same compounds have not been tested in-vivo and in-vitro for potential antiplasmodial activities.